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Optimized paper-based microchip design. This design contains only 4 layers and permits a more homogeneous dispersion of fluid in the device. It can comport 100 μL of sample without leaking. These images are presented in an exploded view to ease visualization and represent experimental data. Original digitalization of the experimental results is available in the Supporting Information (Figure S14).
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Paper-based devices are a portable, user-friendly and affordable technology that is one of the best analytical tools for inexpensive diagnostic devices. Three-dimensional microfluidic paper-based analytical devices (3D-μPADs) are an evolution of single layer devices and they permit effective sample dispersion, individual layer treatment, and multip...
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... The designs arrived at via reversible origami μPADs can be made into irreversible μPADs via multiple methods including hybrid origami/double-sided tape glued systems. 30 Patterns of fluidic distribution on the device depend on the design of the paper-based microchip ( Figure S3, Supporting Information), 5 as well as the hydrodynamic resistance and the fluid velocity in a porous matrix, which are essential parameters in fluidic manipulation in μPADs. 31,32 For a detailed discussion on the topic we refer to Elizalde et al. and Fu and Downs. ...
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... Two other optimization designs were tested (Figures S10− S13) that have the same number of layers as the original design. The combination of these designs led to the fully optimized design (Figure 3), which incorporates just four layers and displays homogeneous fluidic dispersion. 24 This design presents a longer hydrophilic path for each channel in the second layer, which in turn led to the same hydrodynamic resistance for all fluidic connections and homogeneous fluidic dispersion in this device. ...
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... can be seen in Figure 3, volumes as small as 40 μL can reach all the spots in the bottom layer of the optimized device. However, a small excess of liquid (65 μL) provides better results with the enzymatic assay. ...
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... first insight is that the rational design of each layer is critical. The design rule is to ensure that channels have the same hydrodynamic resistance for every branch in a single layer (for example, the second layer in Figure 3). 31 While this design rule is inherently for a 2D system, a 3D device is made of 2D structures layered together: an uneven fluidic hydrodynamic resistance in one 2D layer will affect the final output of the entire 3D structure. ...
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... two hydrophilic channels are brought into contact in adjacent layers, this will create a path of smaller hydrodynamic resistance, as observed in Figure 2, analogous to a short-circuit in an electrical circuit. 35 Hydrophilic channels of adjacent layers should be connected just when the fluid has fully completed the first layer, as shown in Figure 3 (with obvious exclusions for cases in which this is desirable, such as in a flow divider). 37 The third insight is also specific to 3D-μPADs. ...
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... The third insight is also specific to 3D-μPADs. Each layer of the device should maximize usage of the material, such as the second layer of the device depicted in Figure 3. This is due to the fact that the effectiveness of layer assembling is layer- dependent: A higher number of layers diminishes the efficacy of the layer assembling method, resulting in a fewer number of functional devices, 5 or requires more steps in assembly of irreversibly bound 3D-μPADs ( Figure S22). ...
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Citations
... To overcome this issue, Evans et al. [59] reported that a proper selection of the paper support to optimize the flow resistance improves the uniformity of the color distribution. Martinez et al. [60] and Morbioli et al. [61] worked on the optimization reaction area, proposing a diamond shape and an origami geometry, respectively. In particular, in the former work, the authors attributed the improvement to a better dispersion of the colored product. ...
The need for providing rapid and, possibly, on-the-spot analytical results in the case of intoxication has prompted researchers to develop rapid, sensitive, and cost-effective methods and analytical devices suitable for use in nonspecialized laboratories and at the point of need (PON). In recent years, the technology of paper-based microfluidic analytical devices (μPADs) has undergone rapid development and now provides a feasible, low-cost alternative to traditional rapid tests for detecting harmful compounds. In fact, µPADs have been developed to detect toxic molecules (arsenic, cyanide, ethanol, and nitrite), drugs, and drugs of abuse (benzodiazepines, cathinones, cocaine, fentanyl, ketamine, MDMA, morphine, synthetic cannabinoids, tetrahydrocannabinol, and xylazine), and also psychoactive substances used for drug-facilitated crimes (flunitrazepam, gamma-hydroxybutyric acid (GHB), ketamine, metamizole, midazolam, and scopolamine). The present report critically evaluates the recent developments in paper-based devices, particularly in detection methods, and how these new analytical tools have been tested in forensic and clinical toxicology, also including future perspectives on their application, such as multisensing paper-based devices, microfluidic paper-based separation, and wearable paper-based sensors.
... Additionally, the position of the formed interface is dependent on the geometries of patterned paper layer features and proper layer alignment. 42,43 We developed a layer alignment pin tool and protocol to position device features reproducibly and ensure the interface position is only dependent on synchronized delivery of fluid fronts to the detection zone ( Figure S2). This approach consistently provides interfaces at the center of device detection zones ( Figure S1B). ...
The correct interpretation of the result from a point-of-care device is crucial for an accurate and rapid diagnosis and to guide subsequent treatment. Lateral flow tests (LFTs) use a well- established format that was designed to simplify the user experience. However, the LFT device architecture is inherently limited to detecting analytes that can be captured by molecular recognition. Microfluidic paper-based analytical devices (μPADs), like LFTs, have the potential to be used in diagnostic applications at the point of care. However, μPADs have not gained significant traction outside of academic laboratories, in part, because they have often demonstrated a lack of homogeneous shape or color in signal outputs, which consequently can lead to inaccurate interpretation of results by users. Here, we demonstrate a new class of μPADs that generate colorimetric signals at the interfaces of converging liquid fronts (i.e., lines) to control where colorimetric signals are formed without relying on capture techniques. We demonstrate our approach by developing assays for three classes of analytes—an ion, an enzyme, and a small molecule—using iron (III), acetylcholinesterase, and lactate, respectively. Additionally, we show these devices have the potential to support multiplexed assays by generating multiple lines in a common readout zone. These results highlight the ability of this new paper-based device architecture to aid the interpretation of assays that create soluble products by using flow to constrain those colorimetric products in a familiar, line-format output.
... Following the World Health Organization's ASSURED Challenge (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable) 13 , recent research efforts have been focusing on culture-independent POC approaches. Economically, the market (which was not certified by peer review) is the author/funder. ...
The transport, distribution, and mixing of microfluidics often require additional instruments, such as pumps and valves, which are not feasible when operated in point-of-care (POC) settings. Here, we present a simple microfluidic pathogen detection system known as Rotation-Chip that transfers the reagents between wells by manually rotating two concentric layers without using external instruments. The Rotation-Chip is fabricated by a simple computer numerical control (CNC) machining process and is capable of carrying out 60 multiplexed reactions with a simple 30-degree or 60-degree rotation. Leveraging superhydrophobic coating, a high fluid transport efficiency of 92.78% is achieved without observable leaking. Integrated with an intracellular fluorescent assay, an on-chip detection limit of 1.8×10 ⁶ CFU/mL is achieved for ampicillin-resistant Escherichia coli (E. coli) , which is similar to our off-chip results. We also develop a computer vision method to automatically distinguish positive and negative samples on the chip, showing 100% accuracy. Our Rotation-Chip is simple, low-cost, high-throughput, and can display test results with a single chip image, ideal for various multiplexing POC applications in resource-limited settings.
... Among these methods, wax printing (directly printing designed wax patterns on paper) is most frequently used in origami microfluidic devices with the advantages of fast processing and low cost. Figure 4 shows the processing procedure of hydrophobic barriers fabricated by wax printing [29]. Due to the printed wax patterns are only attached to the paper's surface, baking at 150 °C for 2 min (depending on the thickness and types of wax) is required to make wax fully permeate through the whole thickness of the paper (Fig. 4c). ...
... However, the wax printing also has some drawbacks: a heat reflow process is usually required after wax printing to ensure wax penetrates the whole thickness of paper; during the heat reflow, wax penetrates freely inside the porous media of paper, so the width of wax barriers will broaden in a nearly uncontrollable manner [39], which leads to the wax printing is not suitable for ultra-high-precision fabrication. Hence, researchers should focus on integrating wax The processing procedure of origami microfluidic devices fabricated with wax printing [29]. a Design of hydrophobic barriers; b Wax printing; c Wax thermal reflow; d Cutting; e Paper folding printing and heat reflow to simplify fabricating procedures of wax printing method; furthermore, researchers should try to control the direction of wax penetration or build the theory model of wax penetration inside paper to improve the precision of wax barriers. ...
... But the accuracy of origami microfluidic devices will be reduced with multiple folded layers. Accordion-folding [29], as shown in Fig. 7b, is a convenient and straightforward folding mechanism. With the accordion-folding method, researchers simply need to align adjacent layers in sequence, and there is no limit on the number of folded layers. ...
In recent years, origami/paper folding art has been applied to more and more scientific research due to the efficiently producing tiny structures at a low cost; inspired by this, origami microfluidics (the combination of origami/paper folding with paper-based microfluidics) has also been proposed. In origami microfluidic devices, three-dimensional fluid paths can be formed directly by folding the paper with pre-deposited hydrophobic barriers to accomplish complex fluid manipulation. Compared with the layer-by-layer approach, paper folding is more convenient and easier to fabricate three-dimensional microchannels and can meet the increasing requirements of microfluidic end users and improve the functionality of paper-based microdevices. This review is trying to introduce the most current developments of origami paper-based microfluidics, focusing on hydrophobic barrier processing, paper folding, and methods to fix the folded paper. By paper folding, the application prospects of paper-based microfluidics in the biomedical field have been expanded; thus, some biomedical applications of origami microfluidics are also briefly reviewed in this study. Finally, the challenges and perspectives of origami microfluidics were discussed in this review.
... The two arms of the lateral channels are of equal length positioned the same distance away from the sample inlet to equalize flow. 42 Spatially positioning the extraction zones at a distance from the separation materials serves two functions: (i) it permits visible inspection of plasma separation for filling and quality and (ii) minimizes sample contamination by liberated hemoglobin because the collection zones are not located directly beneath the separation membranes ( Figure S2). ...
Plasma separation cards represent a viable approach for expanding testing capabilities away from clinical settings by generating cell-free plasma with minimal user intervention. These devices typically comprise a basic structure of the plasma separation membrane, unconstrained porous collection pad, and utilize either (i) lateral or (ii) vertical fluidic pathways for separating plasma. Unfortunately, these configurations are highly susceptible to (i) inconsistent sampling volume due to differences in the patient hematocrit or (ii) severe contamination due to leakage of red blood cells or release of hemoglobin (i.e., hemolysis). Herein, we combine the enhanced sampling of our previously reported patterned dried blood spot cards with an assembly of porous separation materials to produce a patterned dried plasma spot card for direct processing and storage of cell-free plasma. Linking both vertical separation and lateral distribution of plasma yields discrete plasma collection zones that are spatially protected from potential contamination due to hemolysis and an inlet zone enriched with blood cells for additional testing. We evaluate the versatility of this card by quantitation of three classes of analytes and techniques including (i) the soluble transferrin receptor by enzyme-linked immunosorbent assay, (ii) potassium by inductively coupled plasma atomic emission spectroscopy, and (iii) 18S rRNA by reverse transcriptase quantitative polymerase chain reaction. We achieve quantitative recovery of each class of analyte with no statistically significant difference between dried and liquid reference samples. We anticipate that this sampling approach can be applied broadly to improve access to critical blood testing in resource-limited settings or at the point-of-care.
... The two arms of the lateral channels are of equal length positioned the same distance away from the sample inlet to equalize flow. 41 Spatially positioning the extraction zones at a distance from the separation materials serves two functions: (i) it permits visible inspection of plasma separation for filling and quality and (ii) minimizes sample contamination by liberated hemoglobin because the collection zones are not located directly beneath the separation membranes ( Figure S2). ...
Plasma separation cards represent a viable approach for expanding testing capabilities away from clinical settings by generating cell-free plasma with minimal user intervention. These devices typically comprise a basic structure of plasma separation membrane (PSM), unconstrained porous collection pad, and utilize either (i) lateral or (ii) vertical fluidic pathways for separating plasma. Unfortunately, these configurations are highly susceptible to (i) inconsistent sampling volume due to differences in patient hematocrit or (ii) severe contamination due to leakage of red blood cells (RBCs) or release of hemoglobin (i.e., hemolysis). Herein, we combine the enhanced sampling of our previously reported patterned dried blood spot (pDBS) cards with an assembly of porous separation materials to produce a patterned dried plasma spot (pDPS) card for direct processing and storage of cell-free plasma. Linking both vertical separation and lateral distribution of plasma yields discrete plasma collection zones that are spatially protected from potential contamination due to hemolysis and an inlet zone enriched with blood cells for additional testing. We evaluate the versatility of this card by quantitation of three classes of analytes and techniques including: (i) soluble transferrin receptor by enzyme-linked immunosorbent assay (ELISA), (ii) potassium by inductively coupled plasma atomic emission spectroscopy (ICP-AES), and (iii) 18S rRNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). We achieve quantitative recovery of each class of analyte with no statistically significant difference between dried and liquid reference samples. We anticipate that this sampling approach can be applied broadly to improve access to critical blood testing in resource limited settings or at the point of care.
... To date, several studies have addressed homogeneous color development: the alternation of the pore sizes of the paper materials, 30 the addition of barriers (particle, poly-(dimethylsiloxane) (PDMS), and wax), 31−33 chemical modifications, 33,34 changing the shape of the detection area, 35 and the device structure (paper-layered design). 1,10,16,36,37 The use of paper with a small pore size and additional barriers decreased the wicking rate, which improved the homogeneity of the color development. 30 The immobilization of the reagents on the substrates prevented the flow of the reagent due to wicking. ...
... 35 The layered design enabled the vertical wicking of the sample into the detection zone or homogeneous sample diffusion based on the same hydrodynamic resistance of the channels for homogeneous color development. 1,10,16,36,37 These studies focused on the homogeneous color development in a single detection area. On the other hand, the characterized additional area ahead of the detection zone at the end of the channel allowed for the pretreatment of the analytes prior to their being detected in the detection zone and the improvement of the homogeneity of the color development by minimizing wicking. ...
The development of low-cost, user-friendly paper-based analytical devices (PADs) that can easily measure target chemicals is attracting attention. However, most PADs require manipulation of the sample using sophisticated micropipettes for quantitative analyses, which restricts their user-friendliness. In addition, immobilization of detection molecules to cellulose fibers is essential for achieving good measuring ability as it ensures the homogeneity of color development. Here, we have described a dip-type PAD that does not require pipette manipulation for sample introduction and immobilization of detection molecules to cellulose fibers and its application to ascorbic acid (AA) and pH assays. The PAD consisted of a dipping area and two channels, each with two detection zones. The developed PADs show color distribution in the two detection zones depending on the sample flow from the dipping area. In comparison with a PAD that has one detection zone at the end of the channel, our developed device achieved higher sensitivity (limit of detection (LOD), 0.22 mg/mL) and reproducibility (maximum coefficient of variation (CV), 2.4%) in AA detection. However, in pH detection, the reproducibility of the PAD with one detection zone at the end of the channel (maximum CV, 21%) was worse than that with two zones (maximum CV, 11%). Furthermore, a dipping time over 3 s did not affect color formation or calibration curves in AA detection: LODs at 3 and 30 s dipping time were 18 and 5.8 μg/mL, respectively. The simultaneous determination of AA and pH in various beverages was performed with no significant difference compared to results of the conventional method.
... The design employed for the 3D-lPAD (Fig. 2a) was developed by Morbioli and co-authors [37]. The 3D-lPADs were printed on Whatman No. 1 filter paper using wax toner (Genuine Xerox Solid Black Ink) and a wax printer (Colorqube 8580 DN, Xerox) (Fig. 2b). ...
... After complete drying of the spots (~30 min), the 3D paper device was folded along the white lines, aligning the edges of adjacent layers and superimposing one layer onto another. The 3D-lPAD was positioned between a bottom flat metal plate, perforated 16 times in a 4 Â 4 configuration, according to the bottom layer of the 3D paper device, and two flat magnetic stainless steel sheets on the top (Fig. 2f) [37]. ...
Bioanalyses are commonly performed with blood or serum samples. However, these analyses often require invasive and painful blood collection using a needle or finger pricking. Saliva is an alternative and very attractive biological medium for performing clinical analyses, since it contains many types of clinically relevant biomarkers and compounds. Its collection is straightforward and can be achieved in a non-invasive and stress-free way. However, the analytes are frequently present at low concentrations, while the viscosity of whole saliva hinders its analysis using paper devices, especially those with multiple layers (3D-μPADs). This work explores the use of a simple, fast, and low-cost saliva sample pretreatment using a cotton-paper-syringe filtration system, allowing the analysis of saliva samples using multilayer paper devices. The proposed methodology employs the oxidation of glucose and lactate, catalyzed by specific oxidase enzymes, producing hydrogen peroxide. The detection is based on the fluorescence quenching of carbon dots in the presence of hydrogen peroxidase. The concentrations of the analytes showed good linear correlations with the fluorescence quenching, with LODs of 2.60 × 10⁻⁶ and 8.14 × 10⁻⁷ mol L⁻¹ for glucose and lactate, respectively. The proposed method presented satisfactory intra-day and inter-day repeatabilities, with %RSD values in the range 3.82–6.61%. The enzymatic systems proved to be specific for the analytes and the matrix had no significant influence on the glucose and lactate determinations. The proposed methodology was successfully applied to saliva and serum samples and was validated using certified material.
... Fluid velocity control Signal enhancement assay (C-reactive protein) [69] Source pad of different sizes ( # 2DPNs) Fluid velocity control Signal enhancement assay (BSA-biotin) [20] Source pad of different sizes (2DPNs) Fluid velocity control Signal enhancement assay (malaria protein PfHRP2) [70,71] Three-dimensional device Fluid velocity control Enzymatic assay (glucose and protein) [72] Three-dimensional device Fluid velocity control Colorimetric-enzymatic assay (protein) [73] Three-dimensional device Fluid velocity control Immunoassay (malaria protein PfHRP2) [74] Three-dimensional device Fluid velocity control Colorimetric-enzymatic assay (glucose) [75] Vertical paper legs of different lengths (2DPNs) ...
... Through this technology, stepwise assays can be applied to two-dimensional (2D) paper network chips, and reagent flow can be controlled by adjusting the size of the source pad. In contrast, methods of controlling the flow of fluid by stacking paper layers in a three-dimensional structure have been studied [72][73][74][75]77]. These methods use the principle of controlling the direction of fluid movement in the three dimensions by vertically connecting layers with hydrophobic regions ( Figure 7D). ...
Microfluidic paper-based analytical devices (μPADs) have been suggested as alternatives for developing countries with suboptimal medical conditions because of their low diagnostic cost, high portability, and disposable characteristics. Recently, paper-based diagnostic devices enabling multi-step assays have been drawing attention, as they allow complicated tests, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which were previously only conducted in the laboratory, to be performed on-site. In addition, user convenience and price of paper-based diagnostic devices are other competitive points over other point-of-care testing (POCT) devices, which are more critical in developing countries. Fluid manipulation technologies in paper play a key role in realizing multi-step assays via μPADs, and the expansion of biochemical applications will provide developing countries with more medical benefits. Therefore, we herein aimed to investigate recent fluid manipulation technologies utilized in paper-based devices and to introduce various approaches adopting several principles to control fluids on papers. Fluid manipulation technologies are classified into passive and active methods. While passive valves are structurally simple and easy to fabricate, they are difficult to control in terms of flow at a specific spatiotemporal condition. On the contrary, active valves are more complicated and mostly require external systems, but they provide much freedom of fluid manipulation and programmable operation. Both technologies have been revolutionized in the way to compensate for their limitations, and their advances will lead to improved performance of μPADs, increasing the level of healthcare around the world.
... In additional experiments, an alternate distributor design motivated by multi-layer three-dimensional paper analytical devices (3D µPADs), which are devices designed to distribute a given fluid volume into multiple analytical zones 11,12 , was also tested for its ability to enhance the interaction between dried reagents and the rehydrating fluid. This "multi-inlet distributor" reduced the non-reactive area from 70.4% to 46.7% (P < 0.005; N = 3) and therefore significantly underperformed compared to surface distributors (see Supplementary Fig. S3). ...
Spatially uniform reconstitution of dried reagents is critical to the function of paper microfluidic devices. Advancing fluid fronts in paper microfluidic devices drive (convect) and concentrate rehydrated reagents to the edges, causing steep chemical gradients and imperfect mixing. This largely unsolved problem in paper microfluidics is exacerbated by increasing device dimensions. In this article, we demonstrate that mixing of dried reagents with a rehydrating fluid in paper microfluidics may be significantly enhanced by stacking paper layers having different wicking rates. Compared to single-layer paper membranes, stacking reduced the “non-reactive area”, i.e. area in which the reconstituted reagents did not interact with the rehydrating fluid, by as much as 97% in large (8 cm × 2 cm) paper membranes. A paper stack was designed to collect ~0.9 ml liquid sample and uniformly mix it with dried reagents. Applications of this technology are demonstrated in two areas: (i) collection and dry storage of sputum samples for tuberculosis testing, and (ii) salivary glucose detection using an enzymatic assay and colorimetric readout. Maximizing the interaction of liquids with dried reagents is central to enhancing the performance of all paper microfluidic devices; this technique is therefore likely to find important applications in paper microfluidics.
